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METHODS COMMITTEE REPORTS

Committee on Antimicrobial Efficacy Testing

JAMES AGIN, CHAIR

Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH

45214

DANIEL KLEIN, SECRETARY

STERIS Corp., PO Box 147, Saint Louis, MO 63166-0647

JOE M. ASCENZI

Johnson and Johnson, Advanced Sterilization Products, 33

Technology Dr, Irvine, CA 92618

DONNA B. SUCHMANN

MicroBioTest, Inc., 105B Carpenter Dr, Sterling, VA 20164

LYNNE M. SEHULSTER

Centers for Disease Control and Prevention, Epidemiology

and Laboratory Branch, Division of Healthcare Quality

Promotion, Mailstop A-35, 1600 Clifton Rd NE, Atlanta,

GA 30333

CANDACE MCMANUS

U.S. Food and Drug Administration, Center for Devices

and Radiological Health, Office of Compliance, 2910

McGee Way, Olney, MD 20832

JAFRUL HASAN

U.S. Environmental Protection Agency, 701 Mapes Rd,

Fort Meade, MD 20755

TAJAH LYNETTE BLACKBURN

U.S. Environmental Protection Agency/Public Health

Services, 5708 Shadwell Ct, Unit #102, Alexandria, VA

22309

ALLISON RODRIGUEZ

U.S. Food and Drug Administration, Winchester

Engineering and Analytical Center, 109 Holton St,

Winchester, MA 01890

GAYLE MULBERRY

Hilltop Research, Inc., Main and Mill St, Miamiville, OH45147

VIPIN K. RASTOGI, GENERAL REFEREE

U.S. Army Edgewood Chemical Biological Center, E. 3150

Kingscreek St N, Aberdeen Proving Grounds, MD 21010

EDUARDO GOMEZ, SAFETY ADVISOr

Centers for Disease Control and Prevention, Bioterrorism

Rapid Response and Advanced Technology, Mailstop G42,

NCPDCID, 1600 Clifton Rd NE, Atlanta, GA 30333

ROBERT A. LABUDDE, STATISTICAL ADVISOR

Least Cost Formulations, Ltd, 824 Timberlake Dr, Virginia

Beach, VA 23464

Study Director Report

STEPHEN F. TOMASINO, STUDY DIRECTOR

U.S. Environmental Protection Agency, Environmental

Science Center, Office of Pesticide Programs,

Microbiology Laboratory, 701 Mapes Rd, Ft. Meade, MD

20755-5350

Summary

The U.S. Environmental Protection Agency (EPA) has

statutory authority under the Federal Insecticide, Fungicide,

and Rodenticide Act for regulating antimicrobial products

used to control pathogenic microorganisms on inanimate

surfaces. The EPAs regulations specify that product

performance (efficacy) data must be submitted to support the

registration of antimicrobial products, including sporicides,

bearing claims to control microorganisms that pose a threat to

human health. In addition, Homeland Security Presidential

Directive 10 directs the EPA to take the federal lead for

developing specific standards, protocols, and capabilities to

address the risks of contamination following a biological

weapons attack and developing strategies, guidelines, andplans for decontamination of persons, equipment, and

facilities. EPA has taken action to address this directive and

significantly improve the nations ability to treat contaminated

sites and to allow for safe reoccupancy. Developing proven

standard methods for evaluating and testing the effectiveness

of antimicrobial products, such as those used to

decontaminate facilities contaminated in 2001 with spores of

Bacillus anthracis (anthrax), is critical for protecting public

health. To help facilitate EPAs initiatives to improve and

develop test methods for antimicrobials products, EPA

awarded AOAC INTERNATIONAL a multiyear contract in

2007. AOAC will provide services to assist EPA with single

and multilaboratory validation trials, namely the procedural,

technical, analytical, and statistical peer-review support

services for acceptance of study design protocols and

associated data, and the publication of validated methods for

determining disinfectant efficacy, particularlyfor bioterrorism

agents. The EPA is actively seeking input from the

user/stakeholder community such as the Consumer Specialty

Products Association (CSPA) in this effort. Roundtable

discussions at AOAC Annual Meetings have been initiated by

EPA to engage the stakeholder community and to seek

comment on the proposed revisions.

40B METHODS COMMITTEE REPORTS: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009


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Selected Study Director Topics

966.04 Sporicidal Activity of Disinfectants.A

collaborative study to evaluate several proposed

modifications to the Bacillus component of the method was

completed in 2006. Modified Method 966.04 (Method II),

applicable for testing of liquid disinfectants against spores of

B. subtilis on hard surfaces, was approved as a Revised First

Action method (1) and is available on the AOAC OfficialMethods of Analysis (OMA) Website. Publication of the

complete manuscript appears in the J. AOAC Int. (2). In 2006,

the General Referee recommended continuation of the study

to expand scope of modifications to include Clostridium

sporogenes, suture loop carriers, and other surfaces and

product formulations.

A collaborative study designed to evaluate modifications

applicable to liquid and gaseous formulations when tested

against C. sporogenes on hard (porcelain) surfaces was

initiated in 2008. Egg meat medium, the culture medium for

C. sporogenes currently specified in Method 966.04, is no

longer commercially available and finding a suitablereplacement is critical. In addition, the use of a

nonstandardized extract of raw soil as an amendment to egg

meat medium, as stipulated in the method, may result in a

highly variable spore suspension. Cooked meat medium,

commercially available through Becton Dickinson (Franklin

Lakes, NJ) was selected as a replacement due to its broad use

for the culture and maintenance of clostridia and similarity to

egg meat medium (i.e., content, sold as pellets). Manganese

sulfate, shown to be a suitable replacement for soil extract in

the Bacillus collaborative study (2), was evaluated for

Clostridium in an effort to harmonize the sporulation

protocols for both organisms. Eight laboratories participated

in the study. The data have been collected and the study report

is expected to be submitted to the Methods Committee on

Antimicrobial Efficacy Testing by January 2009. Data from

precollaborative studies on modifications to the Clostridium

component of Method 966.04 were published in 2006 (3). The

development and acceptance of a First Action alternate

Method 966.04, which includes the proposed modifications,

is the goal of this project.

In a related project, EPA is generating in-house data to

support modifications to the B. subtilis suture loop

combination. The proposed modifications will be applicable

to liquid formulations when tested against spores ofB. subtilis

on a porous surface (silk and/or polyester loops) and will beconsistent with previously approved modifications to the

method for porcelain carriers. Comparative evaluations of

current and modified procedures are being used to determine

equivalency of spore loading, HCl resistance, and efficacy.

The single-laboratory data will be used to design

multilaboratory precollaborative studies. Test chemicals used

in the efficacy component of the study are sodium

hypochlorite (bleach), glutaraldehyde, and a combination of

peracetic acid and hydrogen peroxide. The precollaborative

studies are targeted for early 2009.

Validation of the Quantitative Three Step Method for

Sporicides.In 2008, the Three Step Method (TSM), a

quantitative procedure for determining the efficacy of liquid

sporicides, was granted First Action status. Based on the data,

the TSM successfully met the statistical parameters for

validation for quantitative test methods. The TSM was

responsive to the change in efficacy of the chemical

treatments and was highly repeatable. The scope of the TSM

validation included testing liquid formulations against spores

ofB. subtilis (a surrogate for virulent strains ofB. anthracis)

on a hard, nonporous surface. Method 966.04 (Method II) was

used as the reference method. The TSM uses 5 5 1 mm

glass coupons to deliver spores into the sporicidal agent

(400 mL) contained in 1.5 mL microcentrifuge tubes,

3 coupons per chemical treatment. Following exposure to the

test chemical and neutralization, spores are removed from the

carriers in 3 fractions by loosely washing (fraction A),

sonication (fraction B), and prolonged agitation and spore

germination (fraction C). Liquid from each fraction is plated

on recovery medium for viable spore enumeration. Control

counts (water control) are compared to the treated counts andthe level of efficacy is determined by calculating the Log10reduction (LR) of spores; LR = log10 (mean spores/control

carrier) log10 (mean spores/treated carrier). The method was

adopted Official Methods 2008.05 (4). The outcome of the

collaborative study has been published (5). In the near future,

the Study Director will seek to expand the scope of the TSM

protocol (i.e., make minor modifications) to include additional

coupon materials to represent porous surfaces relevant to

buildings and environmental surfaces (e.g., wood, ceiling tile,

concrete). Data (e.g., carrier counts, recovery) on porous

coupons were presented to the Methods Committee on

Antimicrobial Efficacy Testing during the 2008 Annual

Meeting. In addition, the Study Director is interested in

expanding the use of the TSM beyond sporeforming bacteria

to include vegetative bacteria such as Staphylococcus aureus

and Pseudomonas aeruginosa. Preliminary data on this topic

were also presented to the Methods Committee on


Meeting.

Editorial Revisions to OMA Chapter 6

(Disinfectants).The Use-Dilution methods (Methods

955.14, 955.15, 964.02), the Tuberculocidal Activity of

Disinfectants test (Method 965.12), and the Germicidal Spray

Products as Disinfectants test (Method 961.02) have been

prioritized for editorial review. Editorial revisions to theUse-Dilution methods and the Tuberculocidal Activity of

Disinfectants test have been submitted and approved. In 2008,

proposed editorial changes to the Germicidal Spray Products

as Disinfectants test were submitted to the Methods

Committee on Antimicrobial Efficacy Testing. The changes

were discussed during the 2008 Roundtable and included

input previously provided by the CSPA.

Procedural Modifications to Disinfectant Test

Methods.EPA has generated data to support procedural

modifications to the Use-Dilution methods and the

METHODS COMMITTEE REPORTS: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 41B


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Confirmative in vitro Test for Determining Tuberculocidal

Activity (Method 965.12 II).

The AOAC Use-Dilution methods, 955.14 (Salmonella

enterica), 955.15 (Staphylococcus aureus), and 964.02

(Pseudomonas aeruginosa), are used to measure the efficacy

of hospital disinfectants on hard inanimate surfaces. The

methods do notprovide proceduresto assess log density of the

test microbe on inoculated penicylinders (carrier counts).

Without a clear, standardized methodology for measuring andmonitoring of carrier counts, the associated efficacy data may

not be as reliable and repeatable. A report (J. AOAC Int.

manuscript) was submitted to the Methods Committee on

Antimicrobial Efficacy Testing that provides a standardized

procedure to address this issue, and based on carrier count

data collected by 4 laboratories over an 8 year period,

minimum and maximum log density values were proposed to

qualify the test results. In addition, a presentation was made

by the Study Director to the Methods Committee on


Meeting outlining the proposed modifications. Carrier count

data were collected concurrently with performing 242

Use-Dilution tests. The tests were conducted on products

bearing claims against P. aeruginosa and S. aureus with and

without an organic soil load (OSL) added to the inoculum

(depending on the specific product label claim). Six carriers

were assayed per test for a total of 1452 carriers. All 242 mean

log densities were between 6.0 and 7.5 [geometric means

between 1.0 106 and 3.2 107 colony-forming units

(CFU/carrier)]. Across microbes and OSL treatments, the

mean log density (SEM) was 6.7 (0.07) per carrier (a

geometric mean of 5.39 106 CFU/carrier). The mean log

density across 6 carriers per test showed good repeatability

(0.29) and reproducibility (0.32). A minimum mean log

density of 6.0 and a maximum of 7.5 (geometric mean of 3.2107 CFU/carrier) were proposed as validity requirements for

S. aureus and P. aeruginosa. This range provides for the

potential inherent variability that may be experienced across a

wide range of laboratories and the slight effect due to the

addition of an OSL. A follow-up report is planned to present

data to support the carrier count procedure and carrier counts

forS. enterica.

Middlebrook 7H9 (M7H9) agar is the medium specified in

Method 965.12 II for maintaining stock cultures of

Mycobacterium bovis BCG. EPA also uses M7H9 agar plates

for inoculum enumeration. Premade M7H9 is not

commercially available and therefore, preparation requires

valuable time and resources; however, Middlebrook 7H11(M7H11) agar is available commercially and its use as an

alternate growth medium is under investigation. Based on

comparative plate counts and colony morphology, the media

are comparable; thus, M7H11 is an adequate alternate to

M7H9. An official modification to Method 965.12 II will be

pursued with AOAC to allow the use of M7H11 for

maintaining stock cultures and for plating inoculum.

Testing of Towelettes.Standardizing test methodology

for measuring the performance of antimicrobials formulated

as towelette-based products is another key priority. During a

2008 Roundtable discussion, the Study Director and

colleagues discussed the need to review current methods or

pursue the development of a new method for resolving this

issue. EPAs Microbiology Laboratorys Standard Operating

Procedure on testing towelettes was presented as an example

of a method currently in use. Further discussion with the

Methods Committee on Antimicrobial Efficacy Testing and

stakeholders will be necessary to address key methodology

issues such as the type and size of carriers, number of

towelettes per carrier, wiping pattern, measuring surface

wetness, and incubation/recovery of used towelettes.

Call for Methods.AOAC, under contract with the U.S.

EPA, conducted a call for methods in an effort to

verify/validate technology to augment and/or replace standard

plate counts for use in quantitative methodology such as

Method 2008.05 (TSM). The attributes of interest included

(1) easy to use; (2) more efficient than standard plating

techniques with regard to the amount of time spent by the

analyst performing the plating, as well as the resources

required; (3) commercially available; (4) enumeration

medium contains a generic substrate to support the growth ofB. subtilis, S. aureus, P. aeruginosa, and S. enterica;

(5) enumeration medium has a quick turnaround time;

(6) technology for reading results is automated and

commercially available (the capacity to enumerate colonies

using the test kit can be separate from the recovery system);

and (7) enumeration technology provides the ability to store

images of the enumerated medium and its associated data.

Several companies submitted product information to AOAC;

the protocol for down-selecting technologies is under

development and may include the use of the Methods

Committee on Antimicrobial Efficacy Testing or the

formation of an expert review panel to assist in the evaluation.Future Initiatives.During the 2008 Annual Meeting,

several new priorities and topic areas were proposed by the

Study Director. In addition to the towelette test, the new

initiatives include (1) the development of chemical reference

standards for use in collaborative studies and proficiency tests

for antimicrobial methods; (2) the need for specific method

performance criteria for antimicrobial tests; (3) standardized

methodology for testing virucidal products; and

(4) development, standardization, and validation of methods

for biofilm. It is anticipated that each topic area will be further

developed prior to the 2009 Annual Meeting.

The Study Director seeks continued support from AOAC,

the Methods Committee on Antimicrobial Efficacy Testing,

and the stakeholder community in the arena of antimicrobial

test method development and revision.

Committee Actions

The Methods Committee on Antimicrobial Efficacy

Testing, established in April 2007, continues to evaluate

improvements, interpretations, and additions related to

Chapter 6 (Disinfectants) of the OMA.

42B METHODS COMMITTEE REPORTS: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009


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OMA Chapter 6 Editorial Reviews

An effort is underway to update and improve the methods

of Chapter 6 (Disinfectants) of the OMA through editorial

review. Editorial changes to the method, Tuberculocidal

Activity of Disinfectants (965.12) were approved by the

committee. Review of the Germicidal Spray Products as

Disinfectants Test is in progress.

Work Items

(1) Carrier Counts in the Use-Dilution Method.The

EPA requested that the Methods Committee on Antimicrobial

Efficacy Testing consider a proposal to add additional features

to the AOAC Use-Dilution methods (955.15 and 964.02)

designed to enhance the Use-Dilution methods through

standardization of microbial populations on dried inoculated

carriers.

Recommendations

(1) Continue study.

(2) Evaluate intralaboratory and interlaboratoryvariability on carrier counts.

(3) Understand impact of carrier counts on experimental

outcome.

(2) Inquiry into the Addition of a Pellicle Attenuation Step

into the Use-Dilution Method.The Efficacy Working Group

of the CSPA has inquired about a proposal to increase the

reproducibility of the AOAC Use-Dilution methods (955.15

and 964.02) forPseudomonas aeruginosa. This modification

proposes dilution of the broth culture to be used for testing

1:50 in nutrient broth to solubilize any remaining pellicle

fragment and determination of carrier counts following a

3060 min soak in subculture broth.

Recommendations

(1) Continue study.

(2) Examine scientific evidence supporting the change.

(3) Proposed Modification to the Clostridium sporogenes

Portion of the AOAC Sporicidal Activity Test (966.04).A

collaborative study is underway to evaluate proceduralmodifications, including the establishment of a commercially

available culture media, as suggested by EPA to aid in the

conduct and reproducibility of testing C. sporogenes with the

AOAC Sporicidal Activity Test.

Recommendations

(1) Continue study.

(2) Review data generated during collaborative

investigation.

References

(1) Official Methods of Analysis (2005) 18th Ed., AOACINTERNATIONAL, Gaithersburg, MD, Method 966.04

(Method II)

(2) Tomasino, S.F., & Hamilton, M.A. (2006) J. AOAC Int. 89,

13731397

(3) Tomasino, S.F., & Samilot-Friere, L.C. (2007) J. AOAC Int.

90, 825833

(4) Official Methods of Analysis (2005) 18th Ed., AOAC

INTERNATIONAL, Gaithersburg, MD, Method 2008.05

(5) Tomasino, S.F., Pines, R.M., Cottrill, M.P., & Hamilton,

M.A. (2008) J. AOAC Int. 91, 833852

METHODS COMMITTEE REPORTS: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009 43B

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